Project Summary/Abstract Infertility is a major health concern, yet the cause of the infertility of 11% of the men and 21-27% of the women cannot be explained; therefore, there is a clear need to improve the diagnosis of infertility. In terms of treatment, in vitro fertilization (IVF), including intracytoplasmic sperm injection (ICSI) is commonly used, but has been associated with higher risk of preterm birth and low birth weight. To better diagnose and treat infertility, it is important to understand the mechanisms that enable sperm to reach oocytes in the female tract. The female reproductive tract interacts with sperm in many ways, both biochemically and physically. Our long- term objective is to elucidate how physical aspects of the female tract environment influence sperm migration. This project is focused how viscoelasticity of female tract secretions facilitate sperm migration. For example, in humans, semen is naturally deposited at the entrance to the cervix and sperm quickly encounter cervical mucus. It has been shown that sperm swim differently in viscoelastic fluid, as compared with standard sperm medium used in laboratories. Not only does the pattern of flagellar beating change as sperm enter viscoelastic fluid, but as recently reported, sperm form dynamic clusters in viscoelastic fluid. Within the clusters, sperm are oriented in parallel, but are not attached to each other, as reported for some murine species. Our goal is to use microfluidic models to understand how sperm penetrate mucus, as well as how and why they form clusters. Such information would provide insights for infertility diagnosis and treatment. Specific Aim 1 is designed to test the hypothesis that sperm penetration into viscoelastic mucus requires a specific pattern of vigorous motility. A contact line pinning microfluidic based device will be developed to create a wall-less interface between a highly viscoelastic artificial mucus and standard sperm medium. High-speed digital video microscopy will be used to assess and compare swimming speeds, trajectories, and flagellar beating patterns, of sperm before and after entering mucus and to compare the data with that of sperm that contact mucus but fail to enter it. We aim to provide fundamental understanding on what makes sperm capable of penetrating mucus. Specific Aim 2 is focused on understanding the biological implication of the dynamic clustering observed in viscoelastic fluid. The experiments are designed to test the hypothesis that clustering improves sperm capacity to swim against flows of viscoelastic fluid. A microfluidic model will be used to investigate how dynamic clustering of sperm in viscoelastic fluid benefits swimming of sperm against fluid flows. Preliminary data indicate that a higher percentage of sperm participate in clusters, and that larger clusters form, when a fluid flow known to cause upstream orientation of sperm is applied. Information from this research should not only inspire improvement of fertility tests, but also suggest ways to improve selection of sperm for IVF/ICSI.